Labeling of molecules using the perturbed angular correlation of gamma radiation

ABSTRACT

A method of studying molecular motion and orientation by labeling a molecule with a radio active tracer having a half life of an excited state generally comparable to the nuclear relaxation time in this state and measuring the rotational correlation time of a molecule from the perturbed angular correlation.

United States Patent I Baldeschwieler I54] LABELING OF MOLECULES USING THE PERTURBED ANGULAR CORRELATION OF GAMMA RADIATION [72] inventor: John D. Baldeschwieler, 221 Durazno Way, Menlo Park, Calif. 94025 [22] Filed: Nov. 28, 1969 [211 App]. No.: 880,778

[52] US. Cl ..250/7l.5 R, 250/83.3 R, 250/106 T 51 lm. Cl. ..G0lt 1/20 [58] Field oiSearch ..250/71.5 R, 83.3 R, 106T [451 May 23, 1972 Primary Examiner-Archie R. Borchelt Attomey-LimbaCh, Limbach & Sutton ABSTRACT A method of studying molecular motion and orientation by labeling a molecule with a radio active tracer having a half life of an excited state generally comparable to the nuclear relaxa- 1 tion time in this state and measuring the rotational correlation time of a molecule from the perturbed angular correlation.

14 Claims, 10 Drawing Figures 5L w '22 MULTI- wmciiimcc CHANNEL UNIT 1 ANALYZER Patented May 23, 1972 3 Sheets-Sheet 1 C MW 6 5 5 m 0 m .I X m X OJ MM 4 00 7L 2 1. T [TL LL 0 5% 3V Wm 22 2 WV W 07 7 29 4 4 3 Z 247 keV Stable I LII" I MULTI- CHANNEL ANA LVZ ER GATE FAST CDINCI NCE U I T f DET.

FIE- -2- mmocnva AMP DET.

INVENTOR.

Patented May 23, 1972 3,665,192

3 Sheets-Sheet 2 0 so 100 I50 0 so 100 150 t (nsec) t (nsec) FIEr-3A- FIE--35- l 1 0.0- o a 0 50 m0 150 Hnscc) I FIE gg 556 K JOHN 0. BALDESCHW/ELEIZ '"0 50 I50 :30 BY (nsec) 4 FIErA-A-. fiog LABELING OF MOLECULES USING THE PER'IURBED ANGULAR CORRELATION OF GAMMA RADIATION The invention described herein was made in the course of work under a grant or award from the Department of Health, Education, and Welfare.

BACKGROUND OF THE INVENTION A number of labeling techniques have recently been developed for the study of rotational correlation times, internal motions, and conformational changes in biological macromolecules. In one such methd,-the depolarization or decay of fluorescence of small chromophores can be used to measure the rotational correlation time of the chromophore bound to a macromolecule. In another method, the motion and orientation of stable free radicals bound to biomolecules are monitored by electrons in resonance. In a third method, halide ions have been used as chemical probes for nuclear magnetic resonance studies of proteins labeled with metal atoms.

These labeling techniques share a number of general features. Information on localized behavior of the macromolecule near the labeling site is usually accessible. In many instances, the labels can be incorporated into interesting regions of the macromolecule by using selective and specific chemical methods. Labels have been bound chemically to substrate or inhibitor molecules which subsequently interact with active regions of enzymes or antibodies.

On the other hand, there is always some uncertainty as to how much the label affects the system being studied. Particularly is this true where free radicals or reactive ions are used for the study.'Moreover, the foregoing labeling techniques lack sensitivity for use in low concentrations or in vivo. Decay of fluorescence techniques require optical transparency for operation which greatly limits its potential use. ESR and NMR techniques demand large scale and expensive equipment for operation.

SUMMARY OF THE INVENTION AND OBJECTS The present invention relates to a method of studying static and dynamic molecular behavior by affixing a radiation emitter to the moving molecule, detecting the perturbed angular correlation and measuring the rotational correlation time to determine the behavior of the molecule.

It is an object of the present invention to provide an improved method of determining molecular movement rapidly using a label which does not influence the molecule to be studied and which may be used in vivo at low concentrations without the requirement of major equipment.

It is a further object of the invention to determine molecular movement orientation, structure, order and conformation by studying perturbed angular correlation.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a chart illustrating the energy level scheme of "led FIG. 2 is a schematic diagram'of the spectrometer which may be used in performing the method of the present invention.

FIG. 3a is a plot of the anistropy, A(t) as a function of delay time, t, in nanoseconds for "'Cd* in 0.5 M NaCl solution buffered at pH 6.1 with 0.1 M phosphate.

FIG. 3b is a plot of the anisotropy for "'Cd in the presence of 3X10 M native carbonic anhydrase in the above buffer solution.

FIG. 3c is a plot of the anisotropy for "'Cd in the presence of 2.5Xl0" M apo-carbonic anhydrase in the buffer solution.

FIGS. 4a thru 4e are plots of anisotropy at various temperatures for "'Cd solutions containing 1M N-benzyliminodiacefic an (NBIDA) u .3 nq.2? Q,

DESCRIPTION OF THE PREFERRED EMBODIMENTS The direction of emission of radiation, such as gamma rays,

from a sample containing radioactive nuclei is generally isotropic. However, when two gamma rays that are emitted successively from a system of three excited states in nuclear termediate state with fluctuating external fields.

By study of the perturbed angular correlation of gamma radiation from a radioactive nucleus, the nuclear relaxation time can be measured to yield the rotational correlation time, 'rc of a molecule to which the radioactive nucleus is bound. The use of radioactive nucleus as a rotational tracer" to label molecules thus offers the possibility of obtaining the information available with other labeling methods, but with the sensitivity, instrumental simplicity and in vivo applicability of radioactive tracer techniques.

A large number of radioactive nuclei are acceptable. Selection of the proper one depends upon a variety of factors. To measure a nuclear relaxation time, the time a nucleus takes to change its orientation,, using the perturbed angular correlation of gamma radiation, it is desirable to select a radioactive nucleus having a half life in the intermediate nuclear excited state generally comparable. to the nuclear relaxation time. This means that the radioactive nucleus should usually have a relatively long half life and the nuclear electric quadrupole moment in the intermediate state should be large.

For example, the 173-247 keV gamma ray cascade in "Cd can be used for the study of the nuclear relaxation time in the 247 keV state. FIG. 1 shows the cascade in "Cd following electron capture decay of In. As shown in FIG. 1, the energy level scheme of 'Cd also includes a 49 minute metastable state at 397 keV. This state may be populated using the reaction Cd(n,y)"Cd., and nuclear relaxation in the 247 keV state can be studied by using the 150-247 keV cascade. By using this "Cd metastable state, the potentially large recoil effects from the "In electron capture decay are eliminated and only cadmium chemistry is involved.

Examples of other radioactive nuclei having states with convenient properties are shown in the following table, where E is energy of the intermediate state of the cascade measured in keV; T k is the half life of the state; and l is the intrinsic spin characterizingthe state:

TABLE 1 E T Nucleus (keV.) (10* see.) I

' 5 So 68 153 2+ Fe 845 0. 0073 2+ A5 280 o. 24 g;

2 Su 254 22 5- Sn 6. 5 5 C5 81 6. 31 5 2 I13 0. 42 if Table l Continued E T Nucleus (kev.) sec.) v I Hi 9'3 1. 45 2+ Hi 93 1. 53 2 308. ll 0. 081 4 Ta 482 10. 8 i

Us 137 U. 84 2 05 155 0. 73 2* Hg 158 2. 33 i 2 T1 .379 0. 28 {if .2 13b l, 274 .260 4+ Np unn 59.6 0.6 6-

The foregoing table is not exclusive and other nuclei may be used subject to the limitation that half life and nuclear relaxation time be generally comparable. While any one of these nuclei may be used in place of the cadmium system described above, it is preferred to select a nucleus from the group consisting of Sc, Fe, As", Cd", Sn Sn Hg, TI, and Pb because the chemical properties and reactions are well understood and generally useful.

Angular correlation measurements may be made using any suitable apparatus. One apparatus which has been found to be satisfactory, is shown in the schematic diagram of FIG. 2. This is a four-detector fast-slow gamma ray coincidence spectrometer. Spectrometer 11 has four detectors 12, 13, 14 and 15. The detectors may conveniently be made of Na1(Tl) crystals and are arranged at cardinal points around radioactive source 17. The angular coincidence measurements are-made by using various pairs of detectors 12 through 15 at angles of 90 or 180 to the radioactive source17. For example, detector 12 is used to detect a first gamma ray while detector 13 is used to detect a second gamma ray in the cascade in the successive emission in nuclear deexcitation. The slow coincidence unit 18 receives signals from the detectors 12 and 13 through wires 19 and 21, respectively, and discriminates the energies of the gamma rays involved in the cascade. The output pulse from slow coincidence unit 18 proceeds through wire 22 to multi-channel analyzer 23 of known design. The output pulse from slow coincidence unit 18 also gates, through wire 24 to gate 26, the fast side of the spectrometer presently to be described.

The fast coincidence unit 31 also receives the signals from detectors 12 and 13 through wires 19 and 21 respectively. Fast coincidence unit 31 includes a time-to-amplitude converter. This converter sends a pulse through wire 32, gate 26, and wire 33 to multi-channel analyzer 23. The pulse has an amplitude proportional to the delay time between the arrival of the two gamma rays. The multi-channel analyzer 23 then provides a direct display of perturbed angular correlation v. delay time.

In using the aforementioned "Cd system, the coincidence counting rate may be determined as follows. Using the 173-247 keV gamma ray cascade in "Cd following the decay of ln, as shown in FIG. 1, W(0,t) is given by W(0, l) :wm +11 1 (cos 0) J (1 where P is the Legendre polynominal (3 cos61)/ 1- is the mean lifetime of the intermediate nuclear state, t is the time interval between emission of the two gamma rays, and the coefficient A -O.20. For the 247 keV state of Cd, r t /1n 2 1.21.10 seconds. For the 150-247 keV cascade starting from the 49 minute metastable state, Wc0,t) is also given by equation l within A =+O. l 6 l.

The angular correlations of both the l73-247 and l50-247 keV cascades can be perturbed by interaction of the nuclear quadrupole moment of Cd in the 247 keV state with fluctuating electric field gradients at the nucleus. In this case, the

against the delay time t. The shape of this plot depends in detail on the relative magnitude of the molecular rotational correlation time, 1,, and the size of the nuclear quadrupole interaction, eqQ, in the intermediate state.

EXAMPLE I.

FIGS. 3a-c are plots showing the anisotropy as a function of delay time for ""Ca" ion in the presence of native carbonic anhydrase, and "'Ca" ion in the presence of apo-carbonic anhydrase, respectively.

FIG. 3a shows the anisotropy of """Cd in 0.5 M sodium chloride solutions buffered to pH 6.1 with 0.1 M phosphate where the experimental anisotropy A(t) is plotted against the delay time, t, in nanoseconds. The decay of the anisotropy is approximately exponential indicating that the angular correlation is only weakly perturbed, charactieristic of the free ion in solution, for which both eqQ and 1 are small.

FIG. 3b shows the anisotropy for buffered ""Cd in the presence of 3X10 M native carbonic anhydrase. The samples of native enzyme were prepared by adding to 4 ml of the enzyme solution 0.5 ml of a 2X10 M solution of radioactive cadmium chloride. The anisotropy plot is again characteristic of the free ion in solution.

FIG. 3c shows the anisotropy for ""Ca' in the presence of 15x10 M pr n nhys a e i these! b fss. he anisotropy drops sharply from its initial value and exhibits a minimum. The correlation is strongly perturbed. The particular time dependence for A(t) in this case approaches that observed in polycrystalline solids. The shape of the plot suggests that time-independent quadrupole interactions are mainly responsible for the perturbation of the angular correlation.

Under these conditions, the nuclear quadrupole interaction e qQ is much larger than the molecular rotational correlation time, 1 The net result is that the nuclear spin system is effectively immobolized. FIG. 3c then indicates that the cadmium ion is rigidly bound to the apo-enzyme, and that the motion of the metal-enzyme complex is slow compared to l/(e qQ) for Cd in the 247 keV state.

Native carbonic anhydrase was demonstrated to be enzymatically active and was then dialyzed to make apo-carbonic anhydrase. 15 ml of 3x10 4 M carbonic anhydrase in 0.1 M acetate buffer at pH 5.0 was dialyzed for 48 hours against one liter of 10 2 M orthopenanthroline in the same buffer. The orthopenanthroline was removed by dialysis against three oneliter solutions of 0.l M phosphate buffer at pH 6.1 containing 0.5 M NaCl. The concentration of the enzyme was determined spectrophometrically using the extinction coefficient of 4.9 10 M at 280 my). The three samples of each substance were studied, each containing approximately a micromole of cadmium.

EXAMPLE 2.

The radioactive label can be made highly specific by designing chemical complexes which bind tightly to the radioactive nucleus. In many cases, then, these complexes canbe bound chemically to substrate or inhibitor molecules which can be subsequently incorporated into interesting regions of macromolecules. The Cd label can for example be bound with a tight chemical complexing agent such as EDTA which is covalently linked with an active group such as a sulthydryl reagent With this general scheme, concern with the details of of temperature, these plots provide qualitative examples of the behavior of the perturbed angular correlation as a function of the rotational properties of the complex. The plot for the frozen solution is clearly similar to the plot for the ape-enzyme as shown in FIG. 30. The other curves demonstrate that a continuous variation in behavior for the angular correlation may be expected as the molecular rotational correlation time'for the complex changes.

The NBIDA molecule complexes the Cd ion, and also has a functional group which can be modified to react with high specificity at free sulfhydral groups in macromolecules.

In addition to measuring the molecular rotational correlation time as a function of temperature as in Example 2, the radioactive label may be used to observe the behavior of biological macromolecules in vivo. The concentrations of the radioactive label may be as low as molar. For example, precisely when a change in conformation of a molecule can be detected upon changing the external field. Taking pH as an example, if the molecule changes from helix to random coil upon change in pH, study of delayed coincidence spectrum shows at what pH the change takes place. A helical conformation gives a spectrum with a single minimum, whereas a random coil gives a curve with an exponential decay. In addition, the method of the present invention is also used to study the details of molecular motion and conformation of non-biological molecules in solution, surface layers, powders, composite materials, materials on bearing surfaces of moving machinery, etc. Orientation of a labeled molecule is readily determined by analyzing the spectrum. If the plot shows a series of minima, the labeled molecules are generally oriented in the same direction. The spacing of the minima depends on the orienta tion of the principal axis of the array with respect to the direction of the first emission.

The delayed coincidence spectrum of the radio active label in a single crystal or in an ordered array of molecules shows characteristic minima. An ordered array of molecules will give rise to a delayed coincidence spectrum which exhibits .a series of minima. For a randomly orientated array of molecules, only the first of these minima remains. Fibers, tendons and membranes are examples of ordered arrays of molecules that may be studied. Molecular motion, orientation and order are thus conveniently determined by analysis of the delayed coincidence spectrum.

In addition to labeled carbonic anhydrase, as in Example 2, molecular motion and conformation of the following molecules have been satisfactorily studied with rotational tracers: bovine serum albumen (BSA), BSA dimer and poly-lglutamic acid. These molecules may conveniently be studied independently to determine characteristic properties. Then, knowing the characteristic delayed coincidence spectrum exhibited by the labeled molecule, the molecule may be introduced into a larger macromolecule at the area of interest. For example, where structure of a cleft in the macromolecule is to be studied, a rotational tracer of known size may be attached to the macromolecule to determine the tightness of fit" of the rotational tracer within the macromolecule. In this way, the size of the cleft in a macromolecule can be generally determined. Where a smaller rotational tracer is incorporated into the same cleft of the macromolecule, a looser fit" may be reflected by greater movement of rotational tracer as reflected in the delayed coincidence spectra. The net effect of the macromolecule and the rotational tracer may be measured by analysis of the delayed coincidence spectrum. If a single minimum occurs, the attachment of the rotational tracer is rigidly bound to the macromolecule and moves with a rotational correlation time that is long with respect to the inverse of the quadrupolar interaction. Where, on the other hand, the delayed coincidence spectrum exhibits an exponential decay, a loosely bound tracer is indicated.

A large number of labels may be prepared, depending upon the intended purpose. It is preferred to have three elements to the radioactive tracer or label. The first element, of course, is the radioactive emitter, preferably selected from the group contained in Table 1. The second element is a complexing agent to firmly bind the radioactive emitter. The third element is a functional group attached to the chemical complex.

By way of example, complexing agents may be of the porphyrin type or of the ethylene diamine tetraacetic acid type. Acetylacetone, together with derivatives of these complexing agents, are satisfactory. Many other chemical complexing agents are known and any one suitable for complexing with the radioactive emitter may be used.

, Similarly, a wide variety of functional groups may be bound to the complex. As previously indicated, sulfitydryl reagents such as phenyl mercury, are highly satisfactory. These are covalently bound to the complexing agent. Another suitable functional group to be covalently bound to the complex is the iodoacetate group. Enzyme substrates or inhibitors may be also bound as functonal groups to the complex as may alkylating agents and hydrophobic moieties. More broadly, any active group that can conveniently be attached to the complex may be employed as the functional group.

chemical functional group with highly specific reactivity.

Iclaim:

1. In a method of studying molecular behavior the steps of affixing a radiation emitter to the molecule to be studied, said radiation emitter giving two emissions in the same state, detecting the perturbed angular correlation of a cascade of radiation emitted, plotting anisotropy in angular correlation against delay time in the arrival of the two emissions, whereby change in behavior of the molecule is determined.

2. A method as in claim 1, wherein the behavior studied is molecular motion and the rotational correlation time is measured, whereby molecular motion is determined.

3. A method as in claim 1, wherein the behavior studied is molecular orientation in an array of molecules to each of which a radiation emitter is affixed, and measuring the successive minima in the plot of anisotropy versus delay time whereby orientation of the array is determined.

4. A method as in claim 1 wherein the behavior studied is order in an array of molecules to each of which a radiation emitter is affixed, and measuring the order of the successive minima whereby order of the array is determined.

5. A method as in claim 1 wherein the behavior studied is structure and the emitter is first affixed to a label molecule of known properties which, in turn, is associated with the molecule to be studied at the area of structural interest to determine the net effect of the label and molecule under study upon rotational correlation time, whereby the structural characteristics of the molecule are determined.

6. A method as in claim 1 wherein the behavior studied is conformation and the emitter is first affixed to a label molecule of known properties which, in turn, is associated with the molecule to be studied at the area of conformational interest to determine the net effect of the label and molecule under study upon rotational correlation time, whereby the conformational characteristics of the molecule are determined.

7. A method as in claim 1 wherein the radiation emitter is a member of the group consisting of Sc, Fe, As", Cd, Sn", Sn Hg, T1 and Pb.

8. A method as in claim 1 wherein the molecule to be stu- 11. A method as in claim 10 wherein the complexing agent is selected from a group consisting of acetylacetone, ethylene diamine tetraacetic acid, porphyrins and derivatives thereof.

12. A method as in claim 10 wherein the radiation emitter is a member of the group consisting of Se, Fe, As", Cd'", Sn", Sh, Hg T1, and Pb'.

13. A radioactive rotational tracer for measuring rotational correlation time consisting of a radiation emitter giving two emissions in the same state complexed to a complexing agent and having a functional group capable of attaching to a material to be studied.

14. A tracer as in claim 13 wherein the nuclear relaxation time for the state of the radioactive emitter is comparable to its half life in the same state. 

1. In a method of studying molecular behavior the steps of affixing a radiation emitter to the molecule to be studied, said radiation emitter giving two emissions in the same state, detecting the perturbed angular correlation of a cascade of radiation emitted, plotting anisotropy in angular correlation against delay time in the arrival of the two emissions, whereby change in behavior of the molecule is determined.
 2. A method as in claim 1, wherein the behavior studied is molecular motion and the rotational correlation time is measured, whereby molecular motion is determined.
 3. A method as in claim 1, wherein the behavior studied is molecular orientation in an array of molecules to each of which a radiation emitter is affixed, and measuring the successive minima in the plot of anisotropy versus delay time whereby orientation of the array is determined.
 4. A method as in claim 1 wherein the behavior studied is order in an array of molecules to each of which a radiation emitter is affixed, and measuring the order of the successive minima whereby order of the array is determined.
 5. A method as in claim 1 wherein the behavior studied is structure and the emitter is first affixed to a label molecule of known properties which, in turn, is associated with the molecule to be studied at the area of structural interest to determine the net effect of the label and molecule under study upon rotational correlation time, whereby the structural characteristics of the molecule are determined.
 6. A method as in claim 1 wherein the behavior studied is conformation and the emitter is first affixed to a label molecule of known properties which, in turn, is associated with the molecule to be studied at the area of conformational interest to determine the net effect of the label and molecule under study upon rotational correlation time, whereby the conformational characteristics of the molecule are determined.
 7. A method as in claim 1 wherein the radiation emitter is a member of the group consisting of Sc44, Fe56, As75, Cd111, Sn118, Sn120, Hg199, Tl203, and Pb204.
 8. A method as In claim 1 wherein the molecule to be studied is a biological macromolecule in vivo.
 9. A method as in claim 1 wherein the nuclear relaxation time for the state of the radioactive emitter is comparable to its half life in the same state.
 10. In a method of preparing a material to be studied with a radioactive label, the steps of binding a radioactive emitter to a chemical complexing agent having at least one functional group, said emitter giving two emissions in the same state and reacting the functional group with a material to be studied.
 11. A method as in claim 10 wherein the complexing agent is selected from a group consisting of acetylacetone, ethylene diamine tetraacetic acid, porphyrins and derivatives thereof.
 12. A method as in claim 10 wherein the radiation emitter is a member of the group consisting of Sc44, Fe56, As75, Cd111, Sn118, Sh120, Hg199, Tl203, and Pb204.
 13. A radioactive rotational tracer for measuring rotational correlation time consisting of a radiation emitter giving two emissions in the same state complexed to a complexing agent and having a functional group capable of attaching to a material to be studied.
 14. A tracer as in claim 13 wherein the nuclear relaxation time for the state of the radioactive emitter is comparable to its half life in the same state. 